The selective recognition and degradation of mislocalized secretory and membrane proteins has been reconstituted in vitro. In this system, secretory and membrane proteins are synthesized in their non-translocated state simply by omitting ER-derived rough microsomes from the reaction or, if they are included, by inhibiting translocation using cotransin. With either of these manipulations, non-translocated proteins are rapidly ubiquitinated in preparation for their degradation. We are employing both classical fractionation and affinity purification approaches to identify the machinery for the selective recognition and degradation of these non-translocated proteins. In the past year, we have successfully reconstituted many of these events in vitro and have used this system to identify factors. We have identified the E2 enzyme involved, and a key chaperone that facilitates degradation. Our current aim is to identify the remaining factors of this pathway and reconstitute the events with purified components. This will pave the way for understanding mechanistically how the pathway operates. In parallel, we are moving into in vivo systems to understand the importance of this degradation pathway in maintaining cellular protein homeostasis.